Functional complementation analysis of yeast bc(1) mutants - A study of the mitochondrial import of heterologous and hybrid proteins

Previous complementation studies with yeast bc(1) mutants, defective in sub unit VII or VIII, using heterologous and hybrid subunits, suggested that th e requirement for import into mitochondria might significantly restrict the scope of this test for compatible proteins. Prediction algorithms indicate that the N-terminal domain of subunit VII contains all known characteristi cs of a mitochondrial targeting signal, whereas in subunit VIII such a sign al is absent from the N-terminal domain, but possibly present in an interna l region of the protein. Despite the fact that the characteristics of a mit ochondrial import signal are found in the N-terminus of all known subunit-V II orthologues, in vitro import experiments show that the protein of human origin is not imported into yeast mitochondria. In vitro import can be rest ored, however, by replacement of the N-terminal part of the human protein b y the N-terminus of the Saccharomyces cerevisiae orthologue, indicating a r equirement for species-specific elements. Similar experiments were performed with subunit VIII and orthologues thereo f, including a hybrid protein in which the N-terminus of the bovine heart o rthologue was replaced by that of S. cerevisiae. The ability of yeast mitoc hondria to import this hybrid protein, in contrast with the bovine subunit- VIII orthologue itself, indicates that for subunit VIII also the N-terminus , in contradiction of theoretical predictions, contributes to the targeting signal, most likely via species-specific elements. Our findings expose the limitations of the currently available criteria for prediction of the presence and location of a mitochondrial targeting seque nce and highlight the necessity of performing separate import studies for i nterpreting complementation studies as long as the species-specific charact eristics of the import signals have not been identified.